21.1 DNA profiling and PCR Flashcards
what is the genome?
all of the genetic material an organism contains
what is a VNTR?
variable number tandem repeats
short sequences of DNA which are repeated many times within introns
what is a STR?
SHORTER sequences of DNA which are repeated many times
trend in the differences in repeats
different people have a different number of repeats so a different satellite pattern
what does DNA profiling do?
allow you to identify an individual and determine familial relationships
when is DNA profiling used?
forensics
family genetics
medical diagnosis
forensics
used to profile blood to link DNA on a crime scene to the DNA of suspects
medical diagnosis
allows you to identify mutations
first step of DNA fragmentation
extract the DNA
but not a lot of DNA
therefore PCR is done to replicate DNA to get more copies
second step of DNA fragmentation
enzymes are used to cut the DNA at specific sites to create different fragments
what enzymes are used to cut up the DNA sample into fragments?
restriction endonuclease
what are the sites at which restriction endonucleases cut at?
the restriction site
what happens to the VNTRs after fragmentation?
they stay in the same positions in the DNA fragments
what technique is used to separate cut fragments of DNA?
electrophoresis
what is gel electropheresis?
technique which utilises the way charged particles can move through a gel medium using an electrical current
what gel is used in electrophoresis?
agarose
how is the current induced on a gel plate?
the wells containing the DNA fragments are found at the negative end
the other side has the positive end
what is the gel immersed in to separate the DNA strands?
in alkali
why does the DNA move up the gel plate?
DNA is negatively charged and is therefore repelled by the negatively charged bottom of the plate upwards
AND
attracted by the positively charged top
how are the DNA fragments separated in electrophoresis?
the smaller fragments move to the positive electrode QUCKER
the larger fragments move to the positive electrode SLOWER
means that larger and smaller fragments are spread out
why is alkali added to the gel?
to turn the DNA into single strands
what type of blotting is done to the DNA strands?
southern blotting
what is southern blotting?
a nylon sheet is used to soak up the data onto a paper
what are the 2 types of DNA probes?
radioactive
fluorescent
how do the DNA probes bind to the DNA?
they are complimentary to the base sequence of the DNA
what is used in electrophoresis to identify DNA fragments?
a DNA ladder
how are the results seen using radioactive probes?
by using an X-ray
how are the results seen using fluorescent probes?
UV light is used and the probes glow
what is the PCR version of DNA?
an artificial replication of DNA from a small sample to produce a large sample
what is a PCR machine called?
a thermal cycler
step 1 in PCR
95 degrees
the hydrogen bonds in the DNA fragment are broken by a high temperature which separates DNA into two strands
step 2 in PCR
55 degrees
the DNA primers anneal to the ends of the separated DNA strands
step 3 in PCR
72 degrees (optimum for polymerase)
DNA polymerase used to attach complimentary nucleotides starting from the DNA primer
DNA polymerase forms phosphodiester bonds between adjacent nucleotides
product of PCR after the first cycle?
four strands of DNA
so 2 DNA molecules
what DNA polymerase is used for the synthesis of the DNA in PCR?
Taq polymerase
why is Taq polymerase thermophilic?
high temperatures cannot denature Taq polymerase hence it is useful in PCR where temperatures are high
allows PCR to continually happen without needing to replace enzymes
what does DNA ligase do?
make phosphodiester bonds
how is PCR done with RNA fragments?
same process as with DNA
how is PCR done with proteins?
as proteins can be positively and negatively charged, a chemical needs to denature the protein to ensure all the proteins have the same charge
what is different about the genomes of different humans?
the NON-CODING regions
VNTRs are many repeats of introns which are unique to humans
allows use to compare the genomes of humans
coding regions are much similar between humans
